Antimicrobial activity of various extracts from various parts of Calophyllum inophyllum L.

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1 Received: Revised on: Accepted: Antimicrobial activity of various extracts from various parts of Calophyllum inophyllum L. Saravanan. R, Dhachinamoorthi. D, Senthilkumar. K and Thamizhvanan. K. ABSTRACT Saravanan. R, Dhachinamoorthi. D, Senthilkumar. K Department of Pharmaceutical Biotechnology, QIS College of Pharmacy, Vengamukkapalam, Ongole, Indi. Thamizhvanan. K Department of Pharmacy, Sree Vidyanikethan College of Pharmacy, A.Rangampet, Tirupati, Andhra Pradesh, India The Indian flora is extensively utilized as source of any drugs mentioned in the traditional systems of medicine. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases. Therefore, researchers are increasingly turning their attention to folk medicine; looking for new leads to develop better drugs against microbial infections. The aim of the current study was to investigate antimicrobial activity of the various extracts of Calophyllum inophyllum L. An ethanol and ethyl acetate extracts of Calophyllum inophyllum L. (Family: Clusiaceae) were evaluated for antimicrobial activity against clinically important bacterial and fungal sp. The results obtained in the present study suggest that the ethanol and ethyl acetate extracts of Calophyllum inophyllum revealed a significant scope to develop a novel broad spectrum of antibacterial and antifungal herbal formulation. Key words: Antimicrobial activity, Calophyllum inophyllum, Clusiaceae, ethanol extract, ethyl acetate extract. INTRODUCTION *For Correspondence: Saravanan. R Department of Pharmaceutical Biotechnology, QIS College of Pharmacy, Ongole, Andhra Pradesh, India Tel.: rssrmcp@yahoo.co.in Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources (Cragg and Newmann, 2001). Plants have provided a source of inspiration for novel drug compounds, as plant derived medicines have made large contributions to human health and well being. Their role is twofold in the development of new drugs: (1) they may become the base for the development of a medicine, a natural blue print for the development of new drugs or; (2) a phytomedicine to be used for the treatment of diseases (Iwu, 1993 and Irobi et al., 1994) The World Health Organisation (WHO) estimated that 80% of the population of developing countries still relies on traditional medicines, mostly plant drugs, for their primary health care needs. Also, modern pharmacopoeia contains at least 25% drugs derived from plants. Calophyllum inophyllum belongs to the family Clusiaceae and is a tree that can grow 8 to 20 meter tall with a broad spreading crown of irregular branches which exudes white latex when bruised (Da silva, 2001). The leaves have opposite arrangements, and are petiolate, thick and shiny with numerous parallel secondary veins (Ito, 2003 and Ito, 2002). The flower is 25 millimetres (0.98 in) wide and occurs in racemose or paniculate inflorescences consisting of 4 to 15 flowers. Flowering can occur yearround, but usually two distinct flowering periods are observed, in late spring and in late autumn. The fruit is a purplish black globoid-to-ovoid drupe when mature with a single seed. When ripe, the fruit is wrinkled and its color varies from yellow to brownish-red. This species is globally distributed in the Paleotropics (Ito, 2002). Within India, it is distributed in the coastal regions of Orissa, Andhra Pradesh, Maharashtra, Karnataka, Kerala, Tamil Nadu and the Andamans.

2 MATERIALS AND METHODS Plant material Calophyllum inophyllum L was collected from Kollimalai hills at the west of pachaimalais in Namakkal district of Tamilnadu, India. The fresh leaves, fruit, stem, flower and root were collected separately, washed with tap water, chopped into smaller pieces with a knife and then kept in the shade for 30 days to dry and then crushed using mortar and pestle, then further reduced to powder using electric blender and then stored in airtight closed bottles until required. The powder materials were passed through sieve number 40 and used for further studies extract -1.50gm/lit, Yeast extract-1.50gm/lit) and fungal culture in Potato Dextrose medium (potato infusion 200 g/lit, dextrose 20 g/lit) was incubated according to their instructions. The cultures were maintained by subculture process done once in 30 days. Screening for Antibacterial Properties Antibacterial activities of various plant extracts were tested by cup-plate method with some modifications (James Cappuccino and Natalie Sherman, 2009). The culture plates were prepared by pouring 30 ml of Mueller Hinton Agar medium (Himedia, Mumbai) into sterile petri plates. The inoculums suspension was spread uniformly over the agar medium using L-spreader to get uniform distribution of microorganisms. Using a flamed cork borer, well of 5 mm diameter was made in the media at a distance of 1-2 cm from the periphery of the plates. These plates were labelled and 100 µl of each plant extract (at different concentration of extracts i.e. 100, 200, 300 and 400 µg/100 µl) was added aseptically into the well. Then the plates were incubated for 24 to 48 h at 37 C. The affectivity of these extracts was recorded by measuring the diameter of inhibition zone by Antibiotic zone reader. Triplicate was performed and the experiment was repeated thrice and the average values were recorded. The above result was compared with the zone of inhibition produced by standard antibiotic disc like Tetracycline (30µg/disc- Hi media, Mumbai). Figure 1. Various parts of Calophyllum inophyllum L. plant. Preparation of extract The shade dried (100 g) powder of plant materials (leaves, fruit, stem, flower and root) were filled separately in the thimble and extracted successively with 500 ml each of ethanol and ethyl acetate using a Soxhlet extractor for 48 hours. Each extracts were concentrated at reduced pressure using rotary evaporator. The extracts were stored in airtight vials and kept refrigerated (2-8 C) until further analysis for antimicrobial activity studies. Collection of strains The lyophilized form of different strains of microorganism like Salmonella typhi (MTCC 733), Pseudomonas aeruginosa (MTCC 1934), Staphylococcus aureus (MTCC 3160), Escherichia coli (MTCC 1652), Vibrio cholera (MTCC 3904), Candida albicans (MTCC 183), Aspergillus niger (MTCC 281), Candida tropicalis (MTCC 184), Aspergillus fumigates (MTCC 2550) and Alternaria tenuissima (MTCC 2802) were used for the anti microbial study. The above strains were obtained from Microbial Type of Culture Collection (MTCC), Institute of Microbial technology (IMTECH), Chandigarh. Growth and maintenance of the culture Primary bacterial cultures were prepared by inoculating the lyophilized cultures of bacteria in Nutrient Broth (Peptic digest of animal tissue -5.00gm/lit, Sodium chloride-1.50gm/lit, Beef Screening for Antifungal Properties Antifungal activities of various plant extracts were tested by cup-plate method (James Cappuccino and Natalie Sherman, 2009). The culture plates were prepared by pouring 30 ml of Potato Dextrose Agar medium (Hi-media, Mumbai) into sterile petri plates. The inoculums suspension was spread uniformly over the agar medium using L-spreader to get uniform distribution of microorganism 15. Using a flamed cork borer, well of 5 mm diameter was made in the media at a distance of 1-2 cm from the periphery of the plates. These plates were labelled and 100 µl of each plant extract (at different concentration of extracts i.e. 100, 200, 300 and 400 µg/100 µl) was added aseptically into the well. Then the plates were incubated for 24 to 48 h at 25 C. The affectivity of these extracts was recorded by measuring the diameter of inhibition zone by Antibiotic zone reader. Triplicate was performed and the experiment was repeated thrice and the average values were recorded. The above result was compared with the zone of inhibition produced by standard antifungal disc like Fluconazole (10µg/disc- Hi media, Mumbai). RESULT & DISCUSSION The ethno botanical efficacy of various parts like leaf, fruit, stem, flower and root of ethanol and ethyl acetate extracts against various clinically important pathogenic bacteria and fungal species by cup plate method. Results of the antimicrobial screening of the different concentrations of the extract on the bacterial and fungal sp. were observed. Among treatments, maximum in vitro inhibitions of tested bacteria s were shown in Table 1and fungal species in Table 2. Here ethanol extracts were

3 Table 1. Antibacterial activity of and extracts of leaf, fruit, stem, flower and root of Calophyllum inophyllum by cup-plate method. Plant parts Leaf Fruit Stem Flower Root Standard disc Control solvent used Tetracycline concentration Zone of inhibition in diameter (mm) of Extract Salmonella typhi Pseudomonas aeruginosa Staphylococcus aureus Escherichia coli Vibrio colerae µg/100 µl MTCC 733 MTCC 1934 MTCC 3160 MTCC 1652 MTCC µg/disc more effective and produced more zone of inhibition when compared to ethyl acetate extracts. Although the various parts of the plant differ significantly in their activities against the microorganisms tested. The results were shown that while increases in concentration of extract increased the zone of inhibition of the microorganisms. From the above data is evident that ethanolic and ethyl acetate extracts were more active against both gram positive, gram negative bacterial and fungal organisms. The results of present research highlights, the fact that the organic solvent extracts exhibited greater antimicrobial activity because the antimicrobial principles were either polar or non-polar and they were extracted only through the organic solvent medium (Benkeblia, 2004). This tends to show that the active ingredients of the plant parts are better extracted with alcohol than other solvents (Bustamante and Bauer, 2003). The alcohol extracts contain alkaloids, coumarins and tannins (Okemo, 1996). Coumarins and tannins have antibacterial and antihelminthic properties (Hedberg et al., 1983) found that alcohol was more efficient than ethyl acetate, acetone in extracting phytochemicals from plant materials (Eloff, 1998 and Cowan, 1999). CONCLUSION Plant based antimicrobial compounds have enormous therapeutically potential as they can serve the purpose without any side effects that are often associated with synthetic antimicrobials (Priyanka Vijay and Rekha Vijayvergia, 2010). The genus Calophyllum (Clusiaceae) is composed of about different species confined to the warm humid tropics of the world (Stevens, 1980). Some of these species are frequently employed in folk medicine to treat several injuries (Sartori et al., 1999). Extensive chemical investigation of this genus has resulted in the isolation of a wide variety of natural products, including xanthones, coumarins, biflavonoids, chalcones, benzofurans and triterpenes (Da Silva et al., 2001; Ito et al., 2002, 2003; Oger et al., 2003; Isaias et al., 2004). The results of present study supports the traditional usage of the studied Calophyllum inophyllum plants and suggests that some of the plant extracts possess compounds with antimicrobial properties that can be used as antimicrobial agents in

4 Table 2. Antifungal activity of and extracts of leaf, fruit, stem, flower and root of Calophyllum inophyllum by cup-plate method. concentration Zone of inhibition in diameter (mm) Plant parts solvent used Aspergillus Aspergillus Alternaria of Extract Candida albicans niger Candida tropicalis fumigates tenuissima µg/100 µl MTCC 183 MTCC 281 MTCC 184 MTCC 2550 MTCC Leaf Fruit Stem Flower Root Standard disc Fluconazole 10µg/disc Control new drugs for the therapy of infectious diseases caused by pathogens. REFERENCE Benkeblia N. Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and Garlic (Allium sativum). Lebensmwissu- Technol. 2004; 37: Bustamante B. and Bauer J. Antimicrobial activity of selected Peruvian medicinal plants. J. Ethanopharm. 2003; 88: Cowan MM. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 1999; 12: Cragg, GM, Newman, DJ. Medicinals for the millennia. Ann.NY Acad Sci. 2001; 953: Da Silva K. L., Santos A. R. S., Mattos P. E. O., Yunes R. A., Delle-Monache F., and Cechinel Filho V. Chemical composition and analgesic activity of Calophyllum brasiliense leaves. Therapie. 2001; 56: Eloff JN. Which extractan should be used for screening and isolation of antimicrobial components from plants. J. Ethnopharm.1998; 60: 1-8. Hedberg I, Hedberg O, Madati P, Mshigeni KE, Mshiu EN, Samuelsson G. Inventory of plants used in traditional medicine in Tanzania.II. Plants of the family Dilleniaceae to Opiliaceae. J. Ethnopharm.1983; 9: Irobi ON, Moo-Young M, Anderson WA, Daramola, SO. Antimicrobial activity of the bark of Bridelia ferruginea (Euphorbiaceae). Intern. J. Pharmacog.1994; 34: Isaias D. E. B., Niero R., Noldin V. N., Buzzi F. C., Yunes Inst R. A., Delle-Monache F., and Cechinel Filho V.Pharmacological and phytochemical investigations of different parts of Calophyllum brasiliense (Clusiaceae). Die Pharmazie. 2004; 59 (11): Ito C., Itoigawa M., Mishina Y., Cechinel Filho V., Enjo F., Tokuda H., Nishino H., and Furukawa H. Chemical constituents of Calophyllum brasiliense, Structure of three new coumarins and cancer chemo- preventive activity of 4-substituted coumarins. J. Nat. Prod. 2003; 66: Ito C., Itoigawa M., Mishina Y., Cechinel Filho V., Mukainaka T., Tokuda H., Nishino H., and Furukawa H. Chemical constituents of Calophyllum brasiliense: Structure elucidation of seven new xanthones and their cancer chemopreventive activity. J. Nat. Prod.2002; 65:

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